Treatment of biomass to obtain fermentable sugars

ABSTRACT

Biomass is pretreated using a low concentration of aqueous ammonia at high biomass concentration. Pretreated biomass is further hydrolyzed with a saccharification enzyme consortium. Fermentable sugars released by saccharification may be utilized for the production of target chemicals by fermentation.

This application claims the benefit of U.S. Provisional Application No.60/670,437, filed Apr. 12, 2005.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with United States government support underContract No. 04-03-CA-70224 awarded by the Department of Energy. Thegovernment has certain rights in this invention.

FIELD OF THE INVENTION

Methods for treating biomass to obtain fermentable sugars are provided.Specifically, fermentable sugars are obtained by pretreating biomassunder conditions of high solids and low ammonia concentration, followedby saccharification.

BACKGROUND OF THE INVENTION

Cellulosic and lignocellulosic feedstocks and wastes, such asagricultural residues, wood, forestry wastes, sludge from papermanufacture, and municipal and industrial solid wastes, provide apotentially large renewable feedstock for the production of chemicals,plastics, fuels and feeds. Cellulosic and lignocellulosic feedstocks andwastes, composed of carbohydrate polymers comprising cellulose,hemicellulose, glucans and lignin are generally treated by a variety ofchemical, mechanical and enzymatic means to release primarily hexose andpentose sugars, which can then be fermented to useful products.

Pretreatment methods are used to make the carbohydrate polymers ofcellulosic and lignocellulosic materials more readily available tosaccharification enzymes. Standard pretreatment methods havehistorically utilized primarily strong acids at high temperatures;however due to high energy costs, high equipment costs, highpretreatment catalyst recovery costs and incompatibility withsaccharification enzymes, alternative methods are being developed, suchas enzymatic pretreatment, or the use of acid or base at mildertemperatures where decreased hydrolysis of biomass carbohydrate polymersoccurs during pretreatment, requiring improved enzyme systems tosaccharify both cellulose and hemicellulose.

A number of pretreatment methods utilizing base have been proposed.Gould (Biotech. and Bioengr. (1984) 26:46-52) discloses a pretreatmentmethod for lignocellulosic biomass using hydrogen peroxide (H₂O₂). Thetreatment is most efficient using H₂O₂ in an amount of at least 0.25wt/wt with respect to substrate.

Teixeira, L., et al. (Appl. Biochem. and Biotech. (1999) 77-79:19-34)disclosed a series of biomass pretreatments using stoichiometric amountsof sodium hydroxide and ammonium hydroxide, with very low biomassconcentration. The ratio of solution to biomass is 14:1.

Elshafei, A. et al. (Bioresource Tech. (1991) 35:73-80) examined thepretreatment of corn stover utilizing NaOH.

Kim, T. and Y. Lee (Bioresource Technology (2005) 96:2007-2013) reportthe use of high amounts of aqueous ammonia for the pretreatment of cornstover.

Patent Application WO2004/081185 discusses methods for hydrolyzinglignocellulose, comprising contacting the lignocellulose with achemical; the chemical may be a base, such as sodium carbonate orpotassium hydroxide, at a pH of about 9 to about 14, under moderateconditions of temperature, pressure and pH.

U.S. Pat. Nos. 5,916,780 and 6,090,595, describe a pretreatment processwherein a specified ratio of arabinoxylan to total nonstarchpolysaccharides (AX/NSP) is assessed and used to select the feedstock.

In order to be an economically competitive process, a commercial processfor the production of fermentable sugars from a renewable resourcebiomass requires the hydrolysis of carbohydrates in lignocellulosicbiomass to provide high yields of sugars at high concentrations, usinglow amounts of chemicals, to produce a source of fermentable sugars withlow toxicity toward fermentative organisms that convert sugars tovalue-added chemicals and fuels.

SUMMARY OF THE INVENTION

The present invention provides a method for treating biomass to producefermentable sugars. The process of the invention involves a pretreatmentstep wherein biomass at relatively high concentration is treated with alow concentration of ammonia relative to the dry weight of biomass.Following pretreatment, the biomass is treated with a saccharificationenzyme consortium to produce fermentable sugars. In one embodiment ofthe invention, the process comprises:

-   -   a) contacting biomass with an aqueous solution comprising        ammonia to form a biomass-aqueous ammonia mixture, wherein the        ammonia is present at a concentration at least sufficient to        maintain alkaline pH of the biomass-aqueous ammonia mixture but        wherein said ammonia is present at less than about 12 weight        percent relative to dry weight of biomass, and further wherein        the dry weight of biomass is at a high solids concentration of        at least about 15 weight percent relative to the weight of the        biomass-aqueous ammonia mixture; and    -   b) contacting the product of step (a) with a saccharification        enzyme consortium under suitable conditions, to produce        fermentable sugars.

Biomass refers to any cellulosic or lignocellulosic material, forexample, bioenergy crops, agricultural residues, municipal solid waste,industrial solid waste, yard waste, wood, forestry waste andcombinations thereof. The aqueous solution comprising ammonia may bederived from ammonia gas, ammonium hydroxide, urea, and combinationsthereof. According to the process of the invention, the aqueous solutioncomprising ammonia may comprise at least one additional base. Inaddition, according to the process of the invention, vacuum may beapplied to the biomass prior to contacting the biomass with an aqueoussolution comprising ammonia. Ammonia may also be removed prior to step(b); ammonia may be recycled back to the pretreatment reactor. Theammonia and biomass may be reacted in the process of the invention at atemperature that is between about 4° C. and about 200° C. A plasticizer,softening agent or combination thereof may be used in the process of theinvention. In addition, energy may be applied to the biomass before orduring step (a), before or during step (b), or a combination thereof inorder to reduce the size, increase the exposed surface area, and/orincrease the accessability of cellulose, hemicellulose and/oroligosaccharides present in the biomass.

The saccharification enzyme consortium may comprise one or moreglycosidases; the glycosidases may be selected from the group consistingof cellulose-hydrolyzing glycosidases, hemicellulose-hydrolyzingglycosidases, and starch-hydrolyzing glycosidases. Other enzymes in thesaccharification enzyme consortium may include peptidases, lipases,ligninases and feruloyl esterases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the growth of Zymomonas mobilis 8b (described in US PatentApplication Publication 2003/0162271 A1, examples IV, VI and XII) in thepresence or absence of acetamide and acetic acid.

DETAILED DESCRIPTION OF THE INVENTION

Applicants specifically incorporate the entire contents of all citedreferences in this disclosure. Further, when an amount, concentration,or other value or parameter is given as either a range, preferred range,or a list of upper preferable values and lower preferable values, thisis to be understood as specifically disclosing all ranges formed fromany pair of any upper range limit or preferred value and any lower rangelimit or preferred value, regardless of whether ranges are separatelydisclosed. Where a range of numerical values is recited herein, unlessotherwise stated, the range is intended to include the endpointsthereof, and all integers and fractions within the range. It is notintended that the scope of the invention be limited to the specificvalues recited when defining a range.

The present invention provides a process for the treatment of biomass toproduce fermentable sugars; the process involves a pretreatment stepwherein biomass at relatively high concentration is treated with arelatively low concentration of ammonia relative to the dry weight ofthe biomass. The ammonia-treated biomass is then digested with asaccharification enzyme consortium to produce fermentable sugars.

Definitions:

In this disclosure, a number of terms are used. The followingdefinitions are provided:

The term “fermentable sugar” refers to oligosaccharides andmonosaccharides that can be used as a carbon source by a microorganismin a fermentation process.

The term “lignocellulosic” refers to a composition comprising bothlignin and cellulose. Lignocellulosic material may also comprisehemicellulose.

The term “cellulosic” refers to a composition comprising cellulose.

By “dry weight” of biomass is meant the weight of the biomass having allor essentially all water removed. Dry weight is typically measuredaccording to American Society for Testing and Materials (ASTM) StandardE1756-01 (Standard Test Method for Determination of Total Solids inBiomass) or Technical Association of the Pulp and Paper Industry, Inc.(TAPPI) Standard T412 om-02 (Moisture in Pulp, Paper and Paperboard).

The term “target chemical” refers to a chemical produced byfermentation. Chemical is used in a broad sense and includes moleculessuch as proteins, including, for example, peptides, enzymes andantibodies.

A target chemical that is “derivable from biomass” is a target chemicalproduced by a process whereby biomass is hydrolyzed to releasefermentable sugars, and the fermentable sugars are fermented using atleast one biocatalyst to produce a desired target chemical.

The terms “plasticizer” and “softening agent” refer to materials thatcause a reduction in the cohesive intermolecular forces along or betweenpolymer chains. Such materials may act, for example, to decreasecrystallinity, or disrupt bonds between lignin and non-lignincarbohydrate fibers (e.g., cellulose or hemicellulose).

The term “saccharification” refers to the production of fermentablesugars from polysaccharides.

The term “pretreated biomass” means biomass that has been subjected topretreatment prior to saccharification.

“Biomass” refers to any cellulosic or lignocellulosic material andincludes materials comprising cellulose, and optionally furthercomprising hemicellulose, lignin, starch, oligosaccharides and/ormonosaccharides. Biomass may also comprise additional components, suchas protein and/or lipid. According to the invention, biomass may bederived from a single source, or biomass can comprise a mixture derivedfrom more than one source; for example, biomass could comprise a mixtureof corn cobs and corn stover, or a mixture of grass and leaves. Biomassincludes, but is not limited to, bioenergy crops, agricultural residues,municipal solid waste, industrial solid waste, sludge from papermanufacture, yard waste, wood and forestry waste. Examples of biomassinclude, but are not limited to, corn grain, corn cobs, crop residuessuch as corn husks, corn stover, grasses, wheat, wheat straw, barley,barley straw, hay, rice straw, switchgrass, waste paper, sugar canebagasse, sorghum, soy, components obtained from milling of grains,trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes,vegetables, fruits, flowers and animal manure. In one embodiment,biomass that is useful for the invention includes biomass that has arelatively high carbohydrate value, is relatively dense, and/or isrelatively easy to collect, transport, store and/or handle. In oneembodiment of the invention, biomass that is useful includes corn cobs,corn stover and sugar cane bagasse.

For the purposes of this invention, an “aqueous solution comprisingammonia” refers to the use of ammonia gas (NH₃), compounds comprisingammonium ions (NH₄ ⁺) such as ammonium hydroxide or ammonium sulfate,compounds that release ammonia upon degradation such as urea, andcombinations thereof in an aqueous medium.

The concentration of ammonia used in the present method is minimally aconcentration that is sufficient to maintain the pH of thebiomass-aqueous ammonia mixture alkaline and maximally less than about12 weight percent relative to dry weight of biomass. This lowconcentration of ammonia is sufficient for pretreatment, and the lowconcentration may also be less than about 10 weight percent relative todry weight of biomass. A very low concentration of 6 percent ammoniarelative to dry weight of biomass, or less, also may be used forpretreatment. By alkaline is meant a pH of greater than 7.0.Particularly suitable is a pH of the biomass-aqueous ammonia mixturethat is greater than 8. In one embodiment, ammonia is present at lessthan about 10 weight percent relative to dry weight of biomass.Particularly suitable is ammonia at less than about 6 weight percentrelative to dry weight of biomass.

Ammonia as used in the present process provides advantages over otherbases. Ammonia partitions into a liquid phase and vapor phase. Gaseousammonia can diffuse more easily through biomass than a liquid base,resulting in more efficacious pretreatment at lower concentrations.Ammonia also is shown herein in Example 11 to compete with hydrolysis,via ammonolysis, of acetyl esters in biomass to form acetamide.Acetamide is less toxic than acetate to certain fermentation organisms,such as Zymomonas mobilis (as demonstrated herein in Example 12). Thusconversion of acetyl esters to acetamide rather than to acetic acidreduces the need to remove acetic acid. The use of ammonia also reducesthe requirement to supplement growth medium used during fermentationwith a nitrogen source. In addition, ammonia is a low-cost material andthus provides an economical process. Ammonia can also be recycled to thepretreatment reactor during pretreatment or following pretreatment, thusenabling a more economical process. For example, following pretreatment,as the temperature is decreased to that suitable for saccharification,ammonia gas may be released, optionally in the presence of a vacuum, andmay be recycled. In a continuous process, ammonia may be continuouslyrecycled.

According to the present method, the aqueous solution comprising ammoniamay optionally comprise at least one additional base, such as sodiumhydroxide, sodium carbonate, potassium hydroxide, potassium carbonate,calcium hydroxide and calcium carbonate. The at least one additionalbase may be added in an amount that is combined with ammonium to form anamount of total base that is less than about 20 weight percent relativeto dry weight of biomass. Preferably the total second base plus ammoniais in an amount that is less than about 15 weight percent. Additionalbase(s) may be utilized, for example, to neutralize acids in biomass, toprovide metal ions for the saccharification enzymes, or to provide metalions for the fermentation growth medium.

In the present method, the dry weight of biomass is at an initialconcentration of at least about 15% up to about 80% of the weight of thebiomass-aqueous ammonia mixture. More suitably, the dry weight ofbiomass is at a concentration of from about 15% to about 60% of theweight of the biomass-aqueous ammonia mixture. The percent of biomass inthe biomass-aqueous ammonia mixture is kept high to minimize the needfor concentration of sugars resulting from saccharification of thepretreated biomass, for use in fermentation. The high biomassconcentration also reduces the total volume of pretreatment material,making the process more economical.

The biomass may be used directly as obtained from the source, or energymay be applied to the biomass to reduce the size, increase the exposedsurface area, and/or increase the availability of cellulose,hemicellulose, and/or oligosaccharides present in the biomass to ammoniaand to saccharification enzymes used in the second step of the method.Energy means useful for reducing the size, increasing the exposedsurface area, and/or increasing the availability of cellulose,hemicellulose, and/or oligosaccharides present in the biomass to ammoniaand to saccharification enzymes include, but are not limited to,milling, crushing, grinding, shredding, chopping, disc refining,ultrasound, and microwave. This application of energy may occur beforeor during pretreatment, before or during saccharification, or anycombination thereof.

Pretreatment of biomass with ammonia solution is carried out in anysuitable vessel. Typically the vessel is one that can withstandpressure, has a mechanism for heating, and has a mechanism for mixingthe contents. Commercially available vessels include, for example, theZipperclave® reactor (Autoclave Engineers, Erie, Pa.), the Jaygo reactor(Jaygo Manufacturing, Inc., Mahwah, N.J.), and a steam gun reactor((described in General Methods Autoclave Engineers, Erie, Pa.). Muchlarger scale reactors with similar capabilities may be used.Alternatively, the biomass and ammonia solution may be combined in onevessel, then transferred to another reactor. Also biomass may bepretreated in one vessel, then further processed in another reactor suchas a steam gun reactor (described in General Methods; AutoclaveEngineers, Erie, Pa.).

Prior to contacting the biomass with an aqueous solution comprisingammonia, vacuum may be applied to the vessel containing the biomass. Byevacuating air from the pores of the biomass, better penetration of theammonia into the biomass may be achieved. The time period for applyingvacuum and the amount of negative pressure that is applied to thebiomass will depend on the type of biomass and can be determinedempirically so as to achieve optimal pretreatment of the biomass (asmeasured by the production of fermentable sugars followingsaccharification).

The contacting of the biomass with an aqueous solution comprisingammonia is carried out at a temperature of from about 4° C. to about200° C. Initial contact of the biomass with ammonia at 4° C., allowingimpregnation at this temperature, was found to increase the efficiencyof saccharification over non-pretreated native biomass. In anotherembodiment, said contacting of the biomass is carried out at atemperature of from about 75° C. to about 150° C. In still anotherembodiment, said contacting of the biomass is carried out at atemperature of from greater than 90° C. to about 150° C.

The contacting of the biomass with an aqueous solution comprisingammonia is carried out for a period of time up to about 25 hrs. Longerperiods of pretreatment are possible, however a shorter period of timemay be preferable for practical, economic reasons. Typically a period ofammonia contact treatment is about 8 hours or less. Longer periods mayprovide the benefit of reducing the need for application of energy forbreaking-up the biomass, therefore, a period of time up to about 25 hrs.may be preferable.

In one embodiment, the pretreatment process may be performed at arelatively high temperature for a relatively short period of time, forexample at from about 100° C. to about 150° C. for about 5 min to about2 hr. In another embodiment, the pretreatment process may be performedat a lower temperature for a relatively long period of time, for examplefrom about 75° C. to about 100° C. for about 2 hr to about 8 hr. Instill another embodiment, the pretreatment process may be performed atroom temperature (approximately 22-26° C.) for an even longer period oftime of about 24 hr. Other temperature and time combinationsintermediate to these may also be used.

For the pretreatment process, the temperature, time for pretreatment,ammonia concentration, concentration of one or more additional bases,biomass concentration, biomass type and biomass particle size arerelated; thus these variables may be adjusted as necessary to obtain anoptimal product to be contacted with a saccharification enzymeconsortium.

A plasticizer, softening agent, or combination thereof, such as polyols(e.g., glycerol, ethylene glycol), esters of polyols (e.g., glycerolmonoacetate), glycol ethers (e.g., diethylene glycol), acetamide,ethanol, and ethanolamines, may be added in the pretreatment process(i.e., step (a)). A plasticizer may be added as a component of theaqueous ammonia solution, as a separate solution, or as a dry component.

The pretreatment reaction may be performed in any suitable vessel, suchas a batch reactor or a continuous reactor. One skilled in the art willrecognize that at higher temperatures (above 100° C.), a pressure vesselis required. The suitable vessel may be equipped with a means, such asimpellers, for agitating the biomass-aqueous ammonia mixture. Reactordesign is discussed in Lin, K.-H., and Van Ness, H. C. (in Perry, R. H.and Chilton, C. H. (eds), Chemical Engineer's Handbook, 5^(th) Edition(1973) Chapter 4, McGraw-Hill, N.Y.). The pretreatment reaction may becarried out as a batch process, or as a continuous process.

It is well known to those skilled in the art that a nitrogen source isrequired for growth of microorganisms during fermentation; thus the useof ammonia during pretreatment provides a nitrogen source and reduces oreliminates the need to supplement the growth medium used duringfermentation with a nitrogen source. If the pH of the pretreatmentproduct exceeds that at which saccharification enzymes are active, orexceeds the range suitable for microbial growth in fermentation, acidsmay be utilized to reduce pH. The amount of acid used to achieve thedesired pH may result in the formation of salts at concentrations thatare inhibitory to saccharification enzymes or to microbial growth. Inorder to reduce the amount of acid required to achieve the desired pHand to reduce the raw material cost of NH₃ in the present pretreatmentprocess, ammonia gas may be evacuated from the pretreatment reactor andrecycled. Typically, at least a portion of the ammonia is removed, whichreduces the pH but leaves some nitrogen that provides this nutrient foruse in subsequent fermentation.

In order to obtain sufficient quantities of sugars from biomass, thebiomass may be pretreated with an aqueous ammonia solution one time ormore than one time. Likewise, a saccharification reaction can beperformed one or more times. Both pretreatment and saccharificationprocesses may be repeated if desired to obtain higher yields of sugars.To assess performance of the pretreatment and saccharificationprocesses, separately or together, the theoretical yield of sugarsderivable from the starting biomass can be determined and compared tomeasured yields.

Saccharification:

Following pretreatment, the product comprises a mixture of ammonia,partially degraded biomass and fermentable sugars. Prior to furtherprocessing, ammonia may be removed from the pretreated biomass byapplying a vacuum. Removing ammonia lowers the pH, and thus lessneutralizing acid is used to obtain the desired pH for saccharificationand fermentation. This results in a lower salt load in the pretreatmentmixture. Typically some ammonia remains, which is desired to provide anitrogen source for fermentation.

The pretreatment mixture is then further hydrolyzed in the presence of asaccharification enzyme consortium to release oligosaccharides and/ormonosaccharides in a hydrolyzate. Saccharification enzymes and methodsfor biomass treatment are reviewed in Lynd, L. R., et al. (Microbiol.Mol. Biol. Rev. (2002) 66:506-577). In one preferred embodiment, theentire pretreatment mixture comprising both soluble and insolublefractions is utilized in the saccharification reaction.

In another embodiment, prior to saccharification, the aqueous fractioncomprising ammonia and solubilized sugars may be separated frominsoluble particulates remaining in the mixture. Methods for separatingthe soluble from the insoluble fractions include, but are not limitedto, decantation and filtration. The insoluble particulates may berecycled to the pretreatment reactor. The insoluble particulates mayoptionally be washed with an aqueous solvent (e.g., water) to removeadsorbed sugars prior to being recycled to the pretreatment reactor. Theinsoluble fraction may then be subjected to additional treatment withaqueous ammonia solution as described above for pretreatment, followedby saccharification with a saccharification enzyme consortium. Thesoluble fraction may also be concentrated prior to saccharificationusing a suitable process, such as evaporation.

Prior to saccharification, the pretreatment product may be treated toalter the pH, composition or temperature such that the enzymes of thesaccharification enzyme consortium will be active. The pH may be alteredthrough the addition of acids in solid or liquid form. Alternatively,carbon dioxide (CO₂), which may be recovered from fermentation, may beutilized to lower the pH. For example, CO₂ may be collected from afermenter and fed, such as by bubbling, into the pretreatment productwhile monitoring the pH, until the desired pH is achieved. Thetemperature may be brought to a temperature that is compatible withsaccharification enzyme activity, as noted below. Any cofactors requiredfor activity of enzymes used in saccharification may be added.

The saccharification enzyme consortium comprises one or more enzymesselected primarily, but not exclusively, from the group “glycosidases”which hydrolyze the ether linkages of di-, oligo-, and polysaccharidesand are found in the enzyme classification EC 3.2.1.x (EnzymeNomenclature 1992, Academic Press, San Diego, Calif. with Supplement 1(1993), Supplement 2 (1994), Supplement 3 (1995, Supplement 4 (1997) andSupplement 5 [in Eur. J. Biochem. (1994) 223:1-5, Eur. J. Biochem.(1995) 232:1-6, Eur. J. Biochem. (1996) 237:1-5, Eur. J. Biochem. (1997)250:1-6, and Eur. J. Biochem. (1999) 264:610-650, respectively]) of thegeneral group “hydrolases” (EC 3.). Glycosidases useful in the presentmethod can be categorized by the biomass component that they hydrolyze.Glycosidases useful for the present method include cellulose-hydrolyzingglycosidases (for example, cellulases, endoglucanases, exoglucanases,cellobiohydrolases, β-glucosidases), hemicellulose-hydrolyzingglycosidases (for example, xylanases, endoxylanases, exoxylanases,β-xylosidases, arabinoxylanases, mannases, galactases, pectinases,glucuronidases), and starch-hydrolyzing glycosidases (for example,amylases, α-amylases, β-amylases, glucoamylases, α-glucosidases,isoamylases). In addition, it may be useful to add other activities tothe saccharification enzyme consortium such as peptidases (EC 3.4.x.y),lipases (EC 3.1.1.x and 3.1.4.x), ligninases (EC 1.11.1.x), and feruloylesterases (EC 3.1.1.73) to help release polysaccharides from othercomponents of the biomass. It is well known in the art thatmicroorganisms that produce polysaccharide-hydrolyzing enzymes oftenexhibit an activity, such as cellulose degradation, that is catalyzed byseveral enzymes or a group of enzymes having different substratespecificities. Thus, a “cellulase” from a microorganism may comprise agroup of enzymes, all of which may contribute to the cellulose-degradingactivity. Commercial or non-commercial enzyme preparations, such ascellulase, may comprise numerous enzymes depending on the purificationscheme utilized to obtain the enzyme. Thus, the saccharification enzymeconsortium of the present method may comprise enzyme activity, such as“cellulase”, however it is recognized that this activity may becatalyzed by more than one enzyme.

Saccharification enzymes may be obtained commercially, such as Spezyme®CP cellulase (Genencor International, Rochester, N.Y.) and Multifect®xylanase (Genencor). In addition, saccharification enzymes may beproduced biologically, including using recombinant microorganisms.

One skilled in the art would know how to determine the effective amountof enzymes to use in the consortium and adjust conditions for optimalenzyme activity. One skilled in the art would also know how to optimizethe classes of enzyme activities required within the consortium toobtain optimal saccharification of a given pretreatment product underthe selected conditions.

Preferably the saccharification reaction is performed at or near thetemperature and pH optima for the saccharification enzymes. Thetemperature optimum used with the saccharification enzyme consortium inthe present method ranges from about 15° C. to about 100° C. In anotherembodiment, the temperature optimum ranges from about 20° C. to about80° C. The pH optimum can range from about 2 to about 11. In anotherembodiment, the pH optimum used with the saccharification enzymeconsortium in the present method ranges from about 4 to about 10.

The saccharification can be performed for a time of about severalminutes to about 120 hr, and preferably from about several minutes toabout 48 hr. The time for the reaction will depend on enzymeconcentration and specific activity, as well as the substrate used andthe environmental conditions, such as temperature and pH. One skilled inthe art can readily determine optimal conditions of temperature, pH andtime to be used with a particular substrate and saccharificationenzyme(s) consortium.

The saccharification can be performed batch-wise or as a continuousprocess. The saccharification can also be performed in one step, or in anumber of steps. For example, different enzymes required forsaccharification may exhibit different pH or temperature optima. Aprimary treatment can be performed with enzyme(s) at one temperature andpH, followed by secondary or tertiary (or more) treatments withdifferent enzyme(s) at different temperatures and/or pH. In addition,treatment with different enzymes in sequential steps may be at the samepH and/or temperature, or different pHs and temperatures, such as usinghemicellulases stable and more active at higher pHs and temperaturesfollowed by cellulases that are active at lower pHs and temperatures.

The degree of solubilization of sugars from biomass followingsaccharification can be monitored by measuring the release ofmonosaccharides and oligosaccharides. Methods to measure monosaccharidesand oligosaccharides are well known in the art. For example, theconcentration of reducing sugars can be determined using the1,3-dinitrosalicylic (DNS) acid assay (Miller, G. L., Anal. Chem. (1959)31:426-428). Alternatively, sugars can be measured by HPLC using anappropriate column as described herein in the General Methods section.

Fermentable sugars released from biomass can be used by suitablemicroorganisms to produce target chemicals. Following saccharification,but prior to fermentation, the saccharification mixture may beconcentrated by evaporation, for example, to increase the concentrationof fermentable sugars. Optionally, liquid in the saccharificationproduct may be separated from solids in a batch or continuous method.Optionally, the liquid or the entire saccharification product may besterilized prior to fermentation. Depending on the microorganism(s) usedduring fermentation and the pH used during saccharification, the pH maybe adjusted to that suitable for fermentation. In addition, thesaccharification mixture may be supplemented with additional nutrientsrequired for microbial growth. Supplements may include, for example,yeast extract, specific amino acids, phosphate, nitrogen sources, salts,and trace elements. Components required for production of a specificproduct made by a specific biocatalyst may also be included, such as anantibiotic to maintain a plasmid or a cofactor required in an enzymecatalyzed reaction. Also additional sugars may be included to increasethe total sugar concentration. The saccharification mixture may be usedas a component of a fermentation broth, for example, making up betweenabout 100% and about 10% of the final medium.

Temperature and/or headspace gas may also be adjusted, depending onconditions useful for the fermentation microorganism(s). Fermentationmay be aerobic or anaerobic. Fermentation may occur subsequent tosaccharification, or may occur concurrently with saccharification bysimultaneous saccharification and fermentation (SSF). SSF can keep thesugar levels produced by saccharification low, thereby reducingpotential product inhibition of the saccharification enzymes, reducingsugar availability for contaminating microorganisms, and improving theconversion of pretreated biomass to monosaccharides and/oroligosaccharides.

Target chemicals that may be produced by fermentation include, forexample, acids, alcohols, alkanes, alkenes, aromatics, aldehydes,ketones, biopolymers, proteins, peptides, amino acids, vitamins,antibiotics, and pharmaceuticals. Alcohols include, but are not limitedto methanol, ethanol, propanol, isopropanol, butanol, ethylene glycol,propanediol, butanediol, glycerol, erythritol, xylitol, and sorbitol.Acids include acetic acid, lactic acid, propionic acid,3-hydroxypropionic, butyric acid, gluconic acid, itaconic acid, citricacid, succinic acid and levulinic acid. Amino acids include glutamicacid, aspartic acid, methionine, lysine, glycine, arginine, threonine,phenylalanine and tyrosine. Additional target chemicals include methane,ethylene, acetone and industrial enzymes.

The fermentation of sugars to target chemicals may be carried out by oneor more appropriate biocatalysts in single or multistep fermentations.Biocatalysts may be microorganisms selected from bacteria, filamentousfungi and yeast. Biocatalysts may be wild type microorganisms orrecombinant microorganisms, and include Escherichia, Zymomonas,Saccharomyces, Candida, Pichia, Streptomyces, Bacillus, Lactobacillus,and Clostridium. In another embodiment, biocatalysts may be selectedfrom the group consisting of recombinant Escherichia coli, Zymomonasmobilis, Bacillus stearothermophilus, Saccharomyces cerevisiae,Clostridia thermocellum, Thermoanaerobacterium saccharolyticum, andPichia stipitis

Many biocatalysts used in fermentation to produce target chemicals havebeen described and others may be discovered, produced through mutation,or engineered through recombinant means. Any biocatalyst that usesfermentable sugars produced in the present method may be used to makethe target chemical(s) that it is known to produce, by fermentation inthe present method.

Fermentation of carbohydrates to acetone, butanol, and ethanol (ABEfermentation) by solventogenic Clostridia is well known (Jones and Woods(1986) Microbiol. Rev. 50:484-524). A fermentation process for producinghigh levels of butanol, also producing acetone and ethanol, using amutant strain of Clostridium acetobutylicum is described in U.S. Pat.No. 5,192,673. The use of a mutant strain of Clostridium beijerinckii toproduce high levels of butanol, also producing acetone and ethanol, isdescribed in U.S. Pat. No. 6,358,717. Genetically modified strains of E.coli have also been used as biocatalysts for ethanol production(Underwood et al., (2002) Appl. Environ. Microbiol. 68:6263-6272). Agenetically modified strain of Zymomonas mobilis that has improvedproduction of ethanol is described in US 2003/0162271 A1.

Lactic acid has been produced in fermentations by recombinant strains ofE. coli (Zhou et al., (2003) Appl. Environ. Microbiol. 69:399-407),natural strains of Bacillus (US20050250192), and Rhizopus oryzae (Tayand Yang (2002) Biotechnol. Bioeng. 80:1-12). Recombinant strains of E.coli have been used as biocatalysts in fermentation to produce 1,3propanediol (U.S. Pat. No. 6,013,494, U.S. Pat. No. 6,514,733), andadipic acid (Niu et al., (2002) Biotechnol. Prog. 18:201-211). Aceticacid has been made by fermentation using recombinant Clostridia (Cheryanet al., (1997) Adv. Appi. Microbiol. 43:1-33), and newly identifiedyeast strains (Freer (2002) World J. Microbiol. Biotechnol. 18:271-275).Production of succinic acid by recombinant E. coli and other bacteria isdisclosed in U.S. Pat. No. 6,159,738, and by mutant recombinant E. coliin Lin et al., (2005) Metab. Eng. 7:116-127). Pyruvic acid has beenproduced by mutant Torulopsis glabrata yeast (Li et al., (2001) Appl.Microbiol. Technol. 55:680-685) and by mutant E. coli (Yokota et al.,(1994) Biosci. Biotech. Biochem. 58:2164-2167). Recombinant strains ofE. coli have been used as biocatalysts for production ofpara-hydroxycinnamic acid (US20030170834) and quinic acid(US20060003429).

A mutant of Propionibacterium acidipropionici has been used infermentation to produce propionic acid (Suwannakham and Yang (2005)Biotechnol. Bioeng. 91:325-337), and butyric acid has been made byClostridium tyrobutyricum (Wu and Yang (2003) Biotechnol. Bioeng.82:93-102). Propionate and propanol have been made by fermentation fromthreonine by Clostridium sp. strain 17cr1 (Janssen (2004) Arch.Microbiol. 182:482-486). A yeast-like Aureobasidium pullulans has beenused to make gluconic acid (Anantassiadis et al., (2005) Biotechnol.Bioeng. 91:494-501), by a mutant of Aspergillis niger (Singh et al.,(2001) Indian J. Exp. Biol. 39:1136-43). 5-keto-D-gluconic acid was madeby a mutant of Gluconobacter oxydans (Elfari et al., (2005) ApplMicrobiol. Biotech. 66:668-674), itaconic acid was produced by mutantsof Aspergillus terreus (Reddy and Singh (2002) Bioresour. Technol.85:69-71), citric acid was produced by a mutant Aspergillus niger strain(Ikram-Ul-Haq et al., (2005) Bioresour. Technol. 96:645-648), andxylitol was produced by Candida guilliermondii FTI 20037 (Mussatto andRoberto (2003) J. Appl. Microbiol. 95:331-337).4-hydroxyvalerate-containing biopolyesters, also containing significantamounts of 3-hydroxybutyric acid 3-hydroxyvaleric acid, were produced byrecombinant Pseudomonas putida and Ralstonia eutropha (Gorenflo et al.,(2001) Biomacromolecules 2:45-57). L-2,3-butanediol was made byrecombinant E. coli (Ui et al., (2004) Lett. Appl. Microbiol.39:533-537).

Production of amino acids by fermentation has been accomplished usingauxotrophic strains and amino acid analog-resistant strains ofCorynebacterium, Brevibacterium, and Serratia. For example, productionof histidine using a strain resistant to a histidine analog is describedin Japanese Patent Publication No. 8596/81 and using a recombinantstrain is described in EP 136359. Production of tryptophan using astrain resistant to a tryptophan analog is described in Japanese PatentPublication Nos. 4505/72 and 1937/76. Production of isoleucine using astrain resistant to an isoleucine analog is described in Japanese PatentPublication Nos. 38995/72, 6237/76, 32070/79. Production ofphenylalanine using a strain resistant to a phenylalanine analog isdescribed in Japanese Patent Publication No. 10035/81. Production oftyrosine using a strain requiring phenylalanine for growth, resistant totyrosine (Agr. Chem. Soc. Japan 50 (1) R79-R87 (1976), or a recombinantstrain (EP263515, EP332234), and production of arginine using a strainresistant to an L-arginine analog (Agr. Biol. Chem. (1972) 36:1675-1684,Japanese Patent Publication Nos. 37235/79 and 150381/82) have beendescribed. Phenylalanine was also produced by fermentation in Eschericiacoli strains ATCC 31882, 31883, and 31884. Production of glutamic acidin a recombinant coryneform bacterium is described in U.S. Pat. No.6,962,805. Production of threonine by a mutant strain of E. coli isdescribed in Okamoto and Ikeda (2000) J. Biosci Bioeng. 89:87-79.Methionine was produced by a mutant strain of Corynebacterium lilium(Kumar et al, (2005) Bioresour. Technol. 96: 287-294).

Useful peptides, enzymes, and other proteins have also been made bybiocatalysts (for example, in U.S. Pat. No. 6,861,237, U.S. Pat. No.6,777,207, U.S. Pat. No. 6,228,630).

The pretreatment and saccharification of biomass to fermentable sugars,followed by fermentation of the sugars to a target chemical isexemplified in Example 9 herein for the production of ethanol frompretreated corn cobs using Z. mobilis as the biocatalyst for thefermentation of sugars to ethanol. The method of the present inventioncan also be used for the production of 1,3-propanediol from biomass.Biomass undergoes pretreatment and saccharification according to thepresent invention; following (or during) saccharification, E. coli isused to produce 1,3-propanediol as described in Example 10 herein.

Target chemicals produced in fermentation by biocatalysts may berecovered using various methods known in the art. Products may beseparated from other fermentation components by centrifugation,filtration, microfiltration, and nanofiltration. Products may beextracted by ion exchange, solvent extraction, or electrodialysis.Flocculating agents may be used to aid in product separation. As aspecific example, bioproduced 1-butanol may be isolated from thefermentation medium using methods known in the art for ABE fermentations(see for example, Durre, Appl. Microbiol. Biotechnol. 49:639-648 (1998),Groot et al., Process. Biochem. 27:61-75 (1992), and referencestherein). For example, solids may be removed from the fermentationmedium by centrifugation, filtration, decantation, or the like. Then,the 1-butanol may be isolated from the fermentation medium using methodssuch as distillation, azeotropic distillation, liquid-liquid extraction,adsorption, gas stripping, membrane evaporation, or pervaporation.Purification of 1,3-propanediol from fermentation media may beaccomplished, for example, by subjecting the reaction mixture toextraction with an organic solvent, distillation, and columnchromatography (U.S. Pat. No. 5,356,812). A particularly good organicsolvent for this process is cyclohexane (U.S. Pat. No. 5,008,473). Aminoacids may be collected from fermentation medium by methods such asion-exchange resin adsorption and/or crystallization.

EXAMPLES

General Methods and Materials

The following abbreviations are used:

“HPLC” is High Performance Liquid Chromatography, “C” is Centigrade,“kPa” is kiloPascal, “m” is meter, “mm” is millimeter, “kW” is kilowatt,“μm” is micrometer, “μL” is microliter, “mL” is milliliter, “L” isliter, “min” is minute, “mM” is millimolar, “cm” is centimeter, “g” isgram, “kg” is kilogram, “wt” is weight, “hr” is hour, “temp” or “T” istemperature, “theoret” is theoretical, “pretreat” is pretreatment, “DWB”is dry weight of biomass.

Sulfuric acid, ammonium hydroxide, acetic acid, acetamide, yeastextract, 2-morpholinoethanesulfonic acid (MES), potassium phosphate,glucose, xylose, tryptone, sodium chloride and citric acid were obtainedfrom Sigma-Aldrich (St. Louis, Mo.).

Pretreatment Reactors

Zipperclave® Reactor

The 4-liter Zipperclave® reactor (Autoclave Engineers, Erie, Pa.) is abatch pressure vessel equipped with a 2.5-liter Hastelloy® pail for thebiomass charge and an agitator to mix the biomass. The reactor vessel isencircled by an electrical heater controlled at the desired pretreatmenttemperature. Direct steam injection is also used to rapidly bring thebiomass up to pretreatment temperature. Steam pressure is adjusted andcontrolled to maintain the desired pretreatment temperature. Steamcondensate formed by heating the Zipperclave® reactor head plate, vesseland outside of the pail drain to a reservoir formed between the pail andthe inner wall of the reactor to prevent excessive dilution of thepretreated slurry.

Jaygo Reactor

The Jaygo reactor is a 130-liter (approximately 51 cm diameter×91 cmlength), horizontal paddle type reactor (Jaygo Manufacturing, Inc.,Mahwah, N.J.) fabricated of Hastelloy® C-22 alloy. The reactor isequipped with a steam jacket capable of heating to approximately 177° C.(862 kPa). Direct steam injection is also used to rapidly bring thebiomass up to pretreatment temperature. Steam pressure is adjusted andcontrolled to maintain the desired pretreatment temperature. Numerousports allow injection of other solvents and hot liquids.

Steam Gun Reactor Batch Digestion System

The 4-liter steam gun reactor (Autoclave Engineers, Erie, Pa.) is asteam-jacketed reactor consisting of a length of 102 mm schedule 80Hastelloy® pipe closed by two ball valves. Additional electrical heatersare placed on all exposed, non-jacketed surfaces of the reactor andcontrolled to the pretreatment set point temperature. Direct steaminjection is also used to rapidly bring the biomass up to pretreatmenttemperature. Steam pressure is adjusted and controlled to maintain thedesired pretreatment temperature. The bottom of the reactor is neckeddown to 51 mm. All pretreated material exits through a replaceable dieat the bottom of the reactor and is collected in a nylon (Hotfill®) 0.21m³ bag supported within a heavy walled, jacketed, and cooled flash tank.

Disc Refiner

The disc refiner is a Sprout Waldron model 30.5 cm refiner (Andritz,Inc., Muncy, Pa.) equipped with a 11 kW electric motor. The gap betweenthe stationary and rotating plates is variable. The feed auger speed isalso variable, from 0-88 rpm. The inlet to the refiner was modified withsix injection ports to allow the introduction of steam, hot water, orother sweep gases and liquids just ahead of the rotating refiner plate.The refiner was equipped with plates (Durametal, Corp., Tulatin, Oreg.)in either pattern D2A506 in Ni-Hard or pattern 18034-A in Ni-Hard.

Pretreatment and Enzymatic Hydrolysis Reactor (PEHR)

The 9 L PEHReactor (constructed at NREL, Golden, Colo.; see co-pendingUS patent application CL3447) has an approximately 15 cm×51 cm stainlesssteel reaction vessel with an injection lance for introduction ofprocessing reactants. The injection lance is connected using a rotaryjoint to a port in a cover on one end of the vessel, which has anadditional port for vessel access. Four baffles run the length of thevessel wall, and are attached perpendicularly to the wall. The bafflesand twenty-two ceramic attrition media cylinders of 3.2 cm×3.2 cm (E.R.Advanced Ceramics, East Palestine, Ohio), free floating in the vessel,apply mechanical mixing of biomass and reactant as the vessel isrotated, promoting assimilation of reactant into the biomass. ThePEHReactor is placed on a Bellco Cell-Production Roller Apparatus(Bellco Technology, Vineland, N.J.) which provides a mechanism forrotation, and the reactor with roller apparatus is housed in atemperature controlled chamber which provides heat. Vacuum and pressuremay be applied to the reaction vessel by attaching external sources tothe lance-connected port in the cover.

Analytical Methods

Cellulose Quantitation

The amount of cellulose in each starting biomass sample was determinedusing methods well known in the art, such as ASTM E1758-01 “Standardmethod for the determination of carbohydrates by HPLC”.

Measurement of Sugar, Acetamide, Lactic Acid and Acetic Acid Content

Soluble sugars (glucose, cellobiose, xylose, galactose, arabinose andmannose), acetamide, lactic acid and acetic acid in saccharificationliquor were measured by HPLC (Agilent Model 1100, Agilent Technologies,Palo Alto, Calif.) using Bio-Rad HPX-87P and Bio-Rad HPX-87H columns(Bio-Rad Laboratories, Hercules, Calif.) with an appropriate guardcolumns. The sample pH was measured and adjusted to 5-6 with sulfuricacid if necessary. The sample was then passed through a 0.2 μm syringefilter directly into an HPLC vial. The HPLC run conditions were asfollows:

-   -   Biorad Aminex HPX-87P (for carbohydrates):    -   Injection volume: 10-50 μL, dependent on concentration and        detector limits    -   Mobile phase: HPLC grade water, 0.2 μm filtered and degassed    -   Flow rate: 0.6 mL/minute    -   Column temperature: 80-85° C., guard column temperature<60° C.    -   Detector temperature: as close to main column temperature as        possible    -   Detector: refractive index    -   Run time: 35 minute data collection plus 15 minute post run        (with possible adjustment for later eluting compounds)    -   Biorad Aminex HPX-87H (for carbohydrates, acetamide, lactic        acid, acetic acid, and ethanol)    -   Injection volume: 5-10 μL, dependent on concentration and        detector limits    -   Mobile phase: 0.01 N Sulfuric acid, 0.2 μm filtered and degassed    -   Flow rate: 0.6 mL/minute    -   Column temperature: 55° C.    -   Detector temperature: as close to column temperature as possible    -   Detector: refractive index    -   Run time: 25-75 minute data collection        After the run, concentrations in the sample were determined from        standard curves for each of the compounds.

Example 1 Stover Pretreatment at High Biomass Concentration, HighTemperature and Comparison of Ammonia Concentrations

The Zipperclave® reactor vessel and head plate were preheated to thetarget pretreatment temperature before introduction of the biomasscharge by cycling steam into the reactor and venting several times.Condensate formed during preheating was removed by vacuum aspirationbefore pretreatment. The Hastelloy® pail was loaded with 0.635-cm(¼-in.) milled stover (100 g, dry weight basis) and inserted into thepre-warmed reactor. The reactor agitator was set to 20 rpm while avacuum (approximately 85 kPa) was applied to the vessel interior andbiomass charge. Ammonium hydroxide solution of the necessary strength togive a dry weight of biomass concentration of 30 weight percent relativeto the weight of the biomass-aqueous ammonia mixture, as well as thedesired ammonia concentration listed in Table 1, was injected near thebottom of the vessel with a spray type nozzle. Test samples had a finalammonia concentration of 12% relative to dry weight of biomass, whilesamples with a final ammonia concentration of 35% relative to dry weightof biomass were used as a comparison. When the temperature of thebiomass charge reached 50° C., steam was introduced near the bottom ofthe reactor to fluidize and raise the temperature of the biomass chargeto either 140° C. or 170° C. At the end of pretreatment, the reactor wasdepressurized through a vent condenser, and a vacuum (approximately 85kPa) was applied for 3 minutes to lower the temperature and removeadditional ammonia from the pretreated slurry prior to opening thereactor and recovering the pretreated biomass.

Whole, unwashed pretreatment slurry containing 0.5 g of cellulose (basedon initial feedstock composition) was added in a final volume of 50 mLto a 125-mL shake flask. Acetic acid (10-100 μL) was added, to titratethe pH of the ammonia-pretreated biomass to 5.0 before enzyme additionbecause of the sensitivity of the enzymes to high pH environments. ThepH was controlled at 5.0 during saccharification by the addition of 50mM citrate buffer, and the temperature was maintained at 50° C. Spezyme®CP cellulase (Genencor International, Rochester, N.Y.) was added to theconcentration listed for each sample in Table 1. The sugar content ofthe resulting saccharification liquor was determined after 96 hrsaccharification according to the sugar measurement protocol describedin the General Methods. Sugar release after 96 hr is shown in Table 2.Controls for this experiment were 1) untreated corn stover, whichyielded 23% of the theoretical yield of glucose (using 56 mg cellulase/gcellulose) and 2) steam (140° C.) pretreated corn stover, which yielded40% of the theoretical yield of glucose (using 56 mg cellulase/gcellulose); xylose was not measured for the controls.

TABLE 1 Sugar release from pretreated corn stover with 96 hrsaccharification Ammonia Pretreat Cellulase Glucose Xylose (g/100 g TempPretreat (mg/g Release Release DWB) (° C.) Time cellulose) (% theoret)(% theoret) 35 170 5 min 56 68.0 60.0 12 170 5 min 56 58.5 45.3 12 170 5min 11 40.8 27.1 35 140 5 min 56 54.5 41.5 12 140 5 min 56 53.0 31.4 12140 5 min 11 38.9 17.1 12 140 15 min  56 62.4 49.6 12 140 15 min  1141.7 33.6 DWB: dry weight of biomass.

These results indicate that a pretreatment using ammonia at 12% for 15minutes at 140° C., followed by saccharification, releases more glucoseand xylose than when pretreatment is using 35% ammonia for 5 minutes at140° C. Thus, advantages of using lower ammonia can be incorporated by asmall increase in pretreatment time.

Example 2 Stover Pretreatment at High Biomass Concentration, LowTemperature, and Very Low Ammonia

The Jaygo reactor was charged with 0.635-cm milled stover (13 kg, dryweight basis). A vacuum (67.7 kPa) was applied to the vessel, and diluteammonium hydroxide solution was injected to give an ammoniaconcentration of 6.2 g ammonia/100 g dry weight of biomass and a dryweight of biomass concentration 30 weight percent relative to totalweight of the biomass-aqueous ammonia mixture. The vacuum was relieved,and steam was applied to the jacket to heat the stover to 100° C. Thesoaked stover was held at temperature for 8 hr with constant mixing at32 rpm, then allowed to cool overnight with continued mixing of theresulting slurry.

Whole, unwashed pretreatment slurry containing 0.5 g of cellulose (basedon initial feedstock composition) was added in a final volume of 50 mLto a 125-mL shake flask. Acetic acid (10-100 μL) was added, ifnecessary, to titrate the pH of the ammonia-pretreated biomass to 5.0before enzyme addition because of the sensitivity of the enzymes to highpH environments. The pH was controlled at 5.0 during saccharification bythe addition of 50 mM citrate buffer, and the temperature was maintainedat 50° C. Spezyme® CP cellulase (Genencor International, Rochester,N.Y.) was added to 56 mg/g cellulose. The sugar content of the resultingsaccharification liquor was determined after 96 hr saccharificationaccording to the sugar measurement protocol described in the GeneralMethods and is shown in Table 2.

TABLE 2 Sugar release from pretreated corn stover at 96 hr AmmoniaPretreat Cellulase Glucose Xylose (g/100 g Temp Pretreat (mg/g ReleaseRelease DWB) (° C.) Time cellulose) (% theoret) (% theoret) 6.2 100 8 hr56 63.9 44.8

The results indicate that these very low ammonia concentrations and lowtemperature pretreatment conditions, (for a period of 8 hr) are aseffective as using 12% ammonia at 140° C. for 15 min.

Example 3 Cob Pretreatment at High Biomass Concentration, LowTemperature, and Very Low Ammonia Concentration Followed by High BiomassConcentration Saccharification

Whole or fractured corn cobs (approximately 13 kg, dry weight basis)were loaded into the Jaygo reactor. Cobs were fractured by passingthrough the disk refiner (General Methods) equipped with plates C-2975.Resulting fractured cobs were passed through a 1.27 cm screen. Anypieces retained were passed through the disk refiner again with a 0.5 cmsmaller gap. A vacuum was applied to the reactor, and dilute ammoniumhydroxide solution was injected to give the final desired ammoniaconcentration (2% or 6%) and concentration of dry biomass (30% or 40%),as given in Table 3. The vacuum was relieved and steam was applied tothe jacket to heat the cobs while soaking to a temperature of 93° C. forthe whole cob sample and 85° C. for fractured cob samples. Short periodsof increased agitator speeds (up to 96 rpm) were applied in an effort toincrease the heating rate. The soaked cobs were held at temperature for4 or 8 hr with constant mixing at 32 rpm then allowed to cool overnightwith continued mixing.

Before pretreated biomass was removed from the reactor, the reactor wasput under vacuum at 90° C. to strip ammonia out of the pretreatedbiomass. Before saccharification, the pH of the pretreated cob biomasswas adjusted to 5.5 with solid citric acid. About 10 kg of pretreatedwhole cob was saccharified in the Jaygo reactor at 50° C. About 1400 gof pretreated fractured cob was added to the PEHReactor, along with 22ceramic attrition cylinders (3.2 cm diameter×3.2 cm long; E. R. AdvancedCeramics, East Palestine, Ohio), for saccharification. An enzyme mixtureof 28 mg Spezyme CP®/g cellulose in untreated stover plus 28 mg/gcellulose Multifect Xylanase® was used for each saccharificationreaction. The final dry weight of biomass concentration at the beginningof each saccharification was 30% relative to the total weight of thepretreated biomass-saccharification enzyme consortium mixture. ThePEHReactor was rotated axially at 19 rpm while maintaining a temperatureof 50° C. The sugar content of the resulting saccharification liquor wasdetermined according to the sugar measurement protocol in the GeneralMethods. Sugar release after 96 hr is shown in Table 3.

TABLE 3 Sugar release from pretreated corn cobs using a highconcentration of biomass (by dry weight) during saccharification.Glucose Ammonia Release Xylose Pretreat Pretreatment (g/100 g DWB DWB (%Release Feedstock time (hr) temp. (° C.) DWB) (Pretreat) (Saccharif.)theoret) (% theoret) Whole 8 hr 93 6 40% 30% 54.4 46.1 cob Fractured 4hr 85 2 30% 30% 42.1 19.1 cob DWB: dry weight of biomass (percent iscalculated relative to the total weight of the mixture)

Example 4 Cob Pretreatment at High Biomass Concentration, HighTemperature, and Very Low Ammonia Concentration Followed by High BiomassConcentration Saccharification

Fractured corn cobs (13 kg, dry basis) were loaded into the Jaygoreactor. After pulling a vacuum on the reactor, ammonium hydroxidesolution of the proper strength to give 2% ammonia and 30% dry weight ofbiomass concentration was pumped into the reactor with 32 rpm mixing atroom temperature. The contents of the reactor were then heated to 95° C.using low-pressure jacket steam. Once the reactor reached 95° C., directsteam injection was used to heat the contents of the reactor to 145° C.When the reactor reached 145° C., the reactor contents were held at thattemperature for 20 minutes using jacket steam and some direct steaminjection. After 20 minutes, a vacuum was pulled on the vent to thereactor and the shredder motor was turned on for 5 minutes. After 1 hrthe cooling water to the jacket was turned on. The contents of the Jaygoreactor were cooled to between 33° C. and 37° C.; then CO₂ was used topressurize the reactor to 138 kPa. The pressurized CO₂ atmosphere wasmaintained for 30 min. The final temperature of the reactor contents wasbetween 27° C. to 31° C. The pH of the soaked/pretreated biomass wasapproximately 7.5.

Pretreated biomass was removed from the Jaygo reactor and transferred tothe PEHReactor for saccharification at a final dry weight of biomassconcentration at the beginning for saccharification of 30% relative tothe total weight of the pretreated biomass-saccharification enzymeconsortium mixture. The pH was then adjusted to 5.5 with solid citricacid, and the material digested with 28 mg Spezyme CP®/g cellulose and28 mg Multifect Xylanase®/g cellulose in untreated cob as described inExample 3. The sugar content of the resulting saccharification liquorwas determined according to the sugar measurement protocol in theGeneral Methods. The sugar release after 96 hr digestion is shown inTable 4.

TABLE 4 Sugar release from pretreated corn cobs using a highconcentration of biomass (by dry weight) during saccharification.Glucose Xylose Pretreatment Ammonia Release Release Pretreat temperature(g/100 g DWB DWB (% (% Feedstock time (hr) (° C.) DWB) (Pretreat)(Saccharif.) theoret) theoret) Fractured 20 min 145 2 30% 30% 35.9 45.4cob

Example 5 Pretreatment with Addition of Plasticizer

Whole cob was pretreated as described in Example 3 at a dry weight ofbiomass concentration of about 30% relative to the total weight of thebiomass-aqueous ammonia mixture, 2 weight percent ammonia relative todry weight of biomass, 100° C., for 8 hr in the Jaygo reactor with 3weight percent relative to dry weight of biomass of glycerol added toact as a plasticizer. After pretreatment, the pH of the resultingmaterial was adjusted to 5 with solid citric acid. Pretreated cob wasthen digested as described in Example 3. An enzyme mixture of 28 mgSpezyme CP®/g cellulose in untreated stover plus 28 mg/g celluloseMultifect Xylanase® in untreated cob was used. After 96 hr digestion,glucose concentration was 92.3 g/L and xylose concentration was 54.4g/L.

Example 6 Disc Refining of Pretreated Biomass

Stover was pretreated in the manner described in Example 1, withdifferent samples having low ammonia (12%) or comparative ammonia (35%)concentrations, and temperature, time, and enzyme conditions as listedin Table 5. Whole cob was pretreated as described in Example 3 withdifferent samples having very low ammonia (3% or 6%), and otherconditions as listed in Table 5. Following pretreatment, the sampleswere passed through a Sprout Waldron disc refiner. The gap between thestationary plate and rotating plate was set at 0.254 mm (0.010 inch) andthe feed auger speed at 7 rpm. Refined material was saccharified asdescribed in Example 2 and the sugar content of the resultingsaccharification liquor was determined according to the sugarmeasurement protocol in the General Methods. Results of thesaccharification at 96 hr are shown in Table 5. The results showed thatwith disc refining prior to saccharification, better digestibility wasattained, or use of lower enzyme concentrations was effective.

TABLE 5 Digestibility of pretreated material that was disc refined priorto saccharification Xylanase Glucose Xylose Pretreat Ammonia CellulaseAdded Release Release Pretreat Temp (g/100 g (mg/g (mg/g (% (% Feedstocktime (° C.) DWB) cellulose) cellulose) theoret) theoret) Stover 5 min170 35 56 0 87.3 72.5 Stover 5 min 140 35 56 0 76.9 59.4 Stover 5 min170 12 56 0 58.8 39.9 Stover 5 min 170 12 28 28 68.4 62.3 Stover 5 min140 12 56 0 69.1 48.6 Stover 5 min 140 12 11 0 52.6 31.5 Stover 15 min140 12 56 0 61.8 40.1 Stover 15 min 140 12 28 28 66.7 54.1 Stover 8 hr100 6 56 0 79.9 59.6 Cob 8 hr 93 6 56 0 82.4 51.7 Cob 8 hr 93 6 28 2883.1 58.6 Cob 8 hr 100 2 56 0 68.0 38.5 Cob 8 hr 100 2 28 28 81.0 57.3

Example 7 Steam Gun Treatment of Pretreated Biomass

Stover was pretreated as described in Example 1 using conditions of 30%dry weight of biomass relative to total weight of biomass-aqueousammonia mixture, 6 weight percent ammonia relative to DWB, 100° C., 8hr, in the Jaygo reactor. Cob was pretreated as described in Example 3using conditions of 40% dry weight of biomass relative to total weightof biomass-aqueous ammonia mixture, 6 weight percent ammonia relative toDWB, 93° C., 8 hr, in the Jaygo reactor. Samples of each pretreatedbiomass were separately loaded into a 4-liter steam gun reactor.Pretreated material was subjected to 170° C. for 5 min, or 140° C. for20 min before being released through a die. The resulting material wassaccharified as described in Example 2. Results are given in Table 6below. The results showed that steam gun treatment prior tosaccharification improved release of glucose.

TABLE 6 Digestibility of pretreated material after steam gun treatmentSteam Steam Xylanase Glucose Xylose Gun Gun Cellulase Added ReleaseRelease Temp Time (mg/g (mg/g (% (% Feedstock (° C.) (min) cellulose)cellulose) theoret) theoret) Stover 170 5 56 0 77.5 37.5 Stover 170 5 2828 82.5 64.4 Stover 140 20 56 0 73.4 43.4 Stover 140 20 28 28 82.2 66.9Cob 170 5 28 28 70.7 47.4 Cob 170 5 11 11 49.1 38.9 Cob 140 20 28 2855.7 39.0 Cob 140 20 11 11 32.9 24.0

Example 8 Modeling of Pretreatment with Ammonia Recycle

Advantages of ammonia recycle were examined with Aspen models (AspenTechnologies, Cambridge, Mass., version 12.1) for two pretreatmentschemes: low temperature (85° C.), long residence time (4 hr) and hightemperature (130° C.), short residence time (20 min). In each modelthere was a series of three flash tanks operating at successively lowerpressures after the pretreatment reactor to provide a means for ammoniarecycle. As the feed stream entered into each tank, it split into vaporand liquid fractions due to the reduction in pressure. The vaporfraction was recycled to pretreatment, while the liquid fraction went onto the next step in the process. Assuming 2 weight percent ammoniarelative to DWB and approximately 27% dry weight of biomass relative tothe total weight of the biomass-aqueous ammonia mixture in pretreatment,ammonia supplied fresh and from the recycle streams for each process areshown in Table 7. In both models, the flash tanks operated in similarfashion so that ammonia recycle was similar. For both scenarios, morethan half of the required ammonia was supplied through recycle, reducingthe need for and cost of fresh ammonia.

TABLE 7 Ammonia recycle in pretreatment - Aspen model results. HighT/short Low T/long residence time residence time Ammonia Ammonia flowflow rate into Fraction total rate into Fraction total pretreatmentammonia in pretreatment ammonia in (kg/hr) pretreatment (kg/hr)pretreatment Fresh NH₃ 518.6 43.5% 520.2 43.7% From 1^(st) flash 371.231.2% 374.4 31.4% From 2^(nd) flash 137.2 11.5% 135.4 11.4% From 3^(rd)flash 164.1 13.8% 161.2 13.5% Total 1191.2  100% 1191.2  100%

Example 9 Ethanol Production from Low Ammonia-Pretreated andSaccharified Cob Biomass, and Comparison to High Ammonia-Pretreated andSaccharified Stover

Cob hydrolyzate was generated by pretreating whole cobs in the Jaygoreactor for 8 hr at 93° C. with 6 weight percent ammonia relative to dryweight of biomass at a dry weight of biomass concentration of 40 weightpercent relative to the total weight of the biomass-aqueous ammoniamixture, as described in Example 3. After pretreatment, ammonia wasremoved by heating the reactor to 90° C. under vacuum. The pH of thepretreated biomass was then adjusted to 5 with sulfuric acid. Thepretreated biomass was saccharified in the Jaygo reactor at 30% dryweight of biomass relative to the total weight of the pretreatedbiomass-saccharification enzyme consortium mixture with 28 mg/gcellulose Spezyme® cellulase and 28 mg/g cellulose Multifect® xylanasefor 168 hr at 50° C. and pH 5. The resulting hydrolyzate was used forfermentation of Zymomonas mobilis 8b in Sixfors fermentors (INFORS AG,Switzerland). Zymomonas mobilis 8b is a strain of Zymomonas mobilis thathas been genetically engineered to give improved, over wild type,production of ethanol and is described in US Patent ApplicationPublication 2003/0162271 A1 (Examples IV, VI and XII). The cobhydrolyzate comprised 78 g/L glucose, 51 g/L xylose, 6 g/L acetamide,and 7 g/L acetic acid. The cob hydrolyzate was used at 40% and 80%strength, with the balance of the medium being concentrated aqueousmedium consisting of yeast extract, and KH₂PO₄ in quantities such thattheir concentrations in the final slurry were about 5 g/L, and 2 g/Lrespectively. Additionally, in the 40% hydrolyzate slurry, glucose andxylose were added in quantities sufficient to bring their concentrationsto their same levels as in the 80% hydrolyzate slurry. The fermentationwas carried out at 37° C. Agitation in the fermentors was 100 rpm, andpH was maintained at 5.5 by addition of 2 N KOH. The results are shownin Table 8. Sugars and ethanol were analyzed as described in GeneralMethods.

For comparison, stover hydrolyzate was generated by pretreating stoverwith 35 weight percent ammonia relative to dry weight of biomass at adry weight of biomass concentration of about 30 weight percent relativeto the total weight of the biomass-aqueous ammonia mixture at 170° C.for 5 min in the Zipperclave® reactor, as described in Example 1. Thepretreated biomass was enzymatically digested at 30% dry weight ofbiomass relative to the total weight of the pretreatedbiomass-saccharification enzyme consortium mixture with 224 mg/gcellulose Spezyme CP® cellulase at 50° C. and pH 5 to generate a highsugar concentration hydrolyzate for fermentation testing. The resultinghydrolyzate comprised 88 g/L glucose, 52 g/L xylose, 9 g/L acetic acidand 15 g/L lactic acid. For ethanol production, Zymomonas mobilis 8b wasfermented on either 40% or 80% (v/v) hydrolyzate slurry. The remainingvolume was made up of concentrated aqueous medium consisting of yeastextract, KH₂PO₄ and MES buffer in quantities such that theirconcentrations in the final slurry would be about 10 g/L, 2 g/L and 0.1M, respectively. Additionally, in the 40% hydrolyzate slurry, glucoseand xylose were added in quantities sufficient to bring theirconcentrations to the same levels as in the 80% hydrolyzate slurry.Fermentation was done at 30° C. and pH 6 in 25 ml shake flasks with 20ml working volume. Agitation was maintained at 150 rpm. Analysis was asfor the cob hydrolyzate fermentation sample and results are given inTable 8.

TABLE 8 Sugar utilization and ethanol yields in fermentation on cob andstover hydrolyzates. Stover (120 hr endpoint) Cob (72 hr endpoint) 40%80% 40% 80% Hydrolyzate Hydrolyzate Hydrolyzate Hydrolyzate Glucose 100%97% 99% 97% Utilized Xylose utilized 90% 16% 96% 57% Ethanol Yield 77%53% 98% 87%

These results showed that fermentation to produce ethanol from cobhydrolyzate pretreated with low ammonia was more efficient than fromstover pretreated with high ammonia.

Example 10 1-3 Propanediol Production from Very Low Ammonia-Pretreatedand Saccharified Cob Biomass

Hydrolyzate generated from pretreatment and saccharification of cob wasfermented to produce 1,3-propanediol. Hydrolyzate was generated bypretreating cob pieces in the steam gun reactor. First cob biomass wasloaded in the PEHReactor (described in General Methods), a vacuumapplied, and dilute ammonium hydroxide solution was injected to give anammonia concentration of 4 g ammonia/100 g dry weight biomass and a dryweight of biomass concentration of 30 g dry weight of biomass/100 gtotal biomass-aqueous ammonia mixture. The reactor vessel charged withammonia and cob was rotated at 4° C. for 30 min. The contents weretransferred to the steam gun reactor (described in General Methods), thetemperature increased to 145° C., and the mixture was held attemperature for 20 minutes. Material from the steam gun was dischargedinto a flash tank, and vacuum was maintained on the flash tank to aidammonia removal. After pH adjustment, the pretreated biomass wassaccharified at 30 g dry weight of biomass/100 g pretreatedbiomass-saccharification enzyme consortium mixture with 28.4 mg/gcellulose Spezyme CP® cellulase and 10.1 mg active protein/g celluloseenzyme consortium consisting of β-glucosidase, xylanase, β-xylosidaseand arabinofuranosidase for 72 hr at 50° C. and pH 5.5. The resultinghydrolyzate was used as a source of fermentable sugar for conversion to1,3-propanediol by recombinant E. coli strain RJ8n pBE93-k1. Theconstruction of strain RJ8n pBE93-k1 is described in detail in PCTapplication WO/2004/018645 (Example 7), and it is a derivative of strainRJ8n, described in U.S. Pat. No. 6,358,716. The hydrolyzate was used at10% with the balance being aqueous medium consisting of 7.5 g/L KH₂PO₄,2.0 g/L citric acid*H₂O, 4.0 ml/L 28% NH₄OH, 3.0 g/L(NH₄)₂SO₄, 2.0 g/LMgSO₄*7H2O, 0.2 g/L CaCl₂*2H₂O, 0.33 g/L ferric ammonium citrate, 0.5g/L yeast extract, 0.1 mg/L vitamin B12, 1.0 mg/L FeSO4*7H2O, 1 mg/LZnSO4*7H2O, 0.1 g/L CuSO4*5H2O, 1 mg/L CoCl2*6H2O, 0.3 mg/L MnSO4*7H2O,0.1 g/L H₃BO4, 0.10 g/L NaMoO4*2H2O, 10 mg/L NaCl with the final pHadjusted to 6.8. Cultures were started from frozen stocks (15% glycerolas cryoprotectant) in 50 mL of medium in a 250 mL baffled flask. Thecultures were incubated at 34° C. and 300 rpm shaking for 24 hours. Theamount of 1,3-propanediol produced was measured by HPLC under thefollowing conditions:

Column: Showdex SH1011

Sample volume: 20 μL

Mobile phase: 0.01 N H2SO4

Flow rate: 0.5 ml/min

Column temperature 50° C.

Detector: Waters 996 photodiode array

Detector temperature: 40° C.

Run time: 40 min

Results are shown in Table 9 below. Products from glucose fermentationby the RJ8n pBE93-k1 strain of E. coli include glycerol (an intermediatemetabolite) and 1,3-propanediol. Experiments were conducted in duplicateflasks and assayed at 24 hr. In this system, glucose in the hydrolysatewas converted to both glycerol and 1,3-propanediol.

TABLE 9 Substrate utilization and product formation by fermentation withE. coli. Broth at time zero Flask 1, 24 hr Flask 2, 24 hr Glucose (g/L)7.39 2.12 2.10 Glycerol (g/L) 0 3.14 3.14 1,3-propanediol 0 1.03 1.07(g/L) Glucose use 0 71% 72%

Example 11 Formation of Acetamide During Pretreatment

Samples derived from cobs pretreated according to the processesdescribed in Example 3 and Example 4 were analyzed to determine the fateof the acetyl groups in the biomass. The pretreatment liquors(pretreatment mixture with insoluble solids removed) were assayed foracetic acid and acetamide content as follows. The pH of each sample wasadjusted to about 3 with H₂SO₄ (72%). For measurement of acetamide, thesample was passed through a 0.2 μm filter and analyzed by HPLC accordingto the conditions listed below. For measurement of total acetate(includes acetate present as acetic acid and acetamide), the acidifiedsample was autoclaved for 1 hr at 121° C.; acetamide was quantitativelyconverted to acetic acid during this step. After autoclaving, the samplewas allowed to cool. The sample was then passed through a 0.2 μm filterinto a sample vial and analyzed according to the conditions listedbelow.

Acetic acid and acetamide concentrations were determined from standardcurves generated for each.

-   Mobile phase: 0.01 N H₂SO₄, 0.2 μm filtered and degassed-   Flow rate: 0.6 mL/min-   Column temperature: 55-65° C.-   Detector temperature: As close to column temperature as possible-   Detector: Refractive index-   Run time: 60 min-   Column: Biorad Aminex HPX-87H column with corresponding guard column

Results for the 3 different pretreatment conditions assayed are shown inTable 10. In each case, all of the acetyl groups were solubilized toeither acetic acid or acetamide.

TABLE 10 Conversion of acetyl groups in biomass to acetamide duringpretreatment. Fraction of Fraction of Fraction of Pretreat PretreatAmmonia initial acetyl recovered recovered time temp (g/100 g DWBrecovered in acetyl as acetyl as Feedstock (hr) (° C.) DWB) (Pretreat)liquor acetamide acetic acid Whole  8 hr 93 6 40% 100% 44% 56% cobFractured  4 hr 85 2 30% 90% 10% 90% cob Fractured 20 min 145 2 30% 99%9% 91% cob DWB, dry weight of biomass. (percent is calculated relativeto the total weight of the biomass-aqueous ammonia mixture)Using an ammonia concentration of 6%, nearly half of the acetyl groupswere converted to acetamide, which is non-inhibitory for biocatalystgrowth as shown in Example 12.

Example 12 Effect of Acetamide and Acetic acid on Zymomonas Growth

To test the toxicity of acetamide and acetic acid, Z. mobilis strain 8b(described in Example 9) was grown in fermentation medium at pH 6.0 withand without acetamide or acetic acid. Fermentation medium was composedof 10 g/L yeast extract, 2 g/L KH₂PO₄, 70 g/L glucose, 40 g/L xylose and0.1 M MES buffer. Z. mobilis 8b was grown in 25-mL baffled Erlenmeyershake flasks rotating at 150 rpm at 30° C. in unsupplemented medium(control), medium supplemented with 6 g/L acetamide, or mediumsupplemented with 7.2 g/L acetic acid. As shown in FIG. 1, the presenceof acetamide had no influence on the growth rate or final density of Z.mobilis, whereas the presence of acetic acid resulted in a reducedgrowth rate and lower cell yield (as measured by dry cell mass).

Example 13 Pretreatment of Bagasse at High Biomass Concentration, HighTemperature, and Very Low Ammonia, and Saccharification at Low and HighConcentration

The PEHReactor (described in General Methods), with no attrition media,was charged with 1.27 cm-milled bagasse (370 g, dry weight basis). Thissugar cane bagasse was NIST Reference Material RM8491, from sugar caneclone H65-7052, originally obtained from the Hawaii Sugar PlantersAssociation, Kunia substation, Oahu, Hi. It was milled in a Wiley millto pass through a 2 mm screen, with the fines (+74 mesh) removed. ThePEHReactor vessel was cooled to 4° C. by rotation in contact with ice onthe outer surface. A vacuum was applied to the reactor vessel, anddilute ammonium hydroxide solution, that was pre-cooled in a cold roomat 4° C. and passed through tubing immersed in an ice-water bath, wasinjected to give an ammonia concentration of 4 g/100 g dry weight ofbiomass and a dry weight of biomass concentration of 45 g/100 g totalbiomass-aqueous ammonia mixture. The reactor vessel charged with ammoniaand bagasse was cooled to 4° C. by applying ice to the surface of therotating reactor vessel, and rotated at 4° C. for 30 min. At this timethe contents were transferred to the steam gun reactor that is describedin General Methods. Once the steam gun reactor was charged with theammonia-bagasse mixture, the temperature was increased to 145° C. andthe mixture was held at temperature for 20 minutes. At the end of thepretreatment time, the bagasse was discharged from the steam gun reactorthrough a 1-in circular die into a flash tank. A sample of pretreatedbagasse was subsequently saccharified in a shake flask and anothersample (approximately 163 g dry weight) was saccharified in thePEHReactor. The shake flask saccharification was carried out at 5% dryweight of biomass relative to the total weight of the pretreatedbiomass-saccharification enzyme consortium mixture, while the PEHReactorsaccharification was carried out at 30% dry weight of biomass relativeto the total weight of the pretreated biomass-saccharification enzymeconsortium mixture. The temperature was maintained at 50° C.

For the PEHReactor saccharification, about 476 g (˜163 g dry weight)pretreated biomass and 22 ceramic attrition cylinders were added to thereactor vessel. The pH was adjusted to 5.0-5.5 with solid citric acid.The reactor vessel was kept inside an incubator chamber controlled to50° C. and rotated axially at 19 rpm. Unpretreated bagasse was alsosaccharified at 5% dry weight of biomass relative to the total weight ofthe pretreated biomass-saccharification enzyme consortium mixture in ashake flask. All saccharifications were done with 28.4 mg/g celluloseSpezyme CP® cellulase and 28.4 mg/g cellulose Multifect® xylanase at 50°C. and pH 5.5 for 96 hr. Yields given in Table 11 below are the releaseas percent of theoretical yield.

TABLE 11 Yields following pretreatment and saccharification of bagasse.No pretreatment Pretreated Pretreated 5% 5% DWB 30% DWB saccharificationsaccharification saccharification Monomer 0.5% 16.6% 23.3% glucose Totalglucose ND ND 36.4% Monomer 1.3% 15.6% 17.2% xylose Total xylose ND ND37.4% ND: not determined

The results demonstrate that pretreatment of bagasse with very lowammonia allows substantial sugar release as compared to the unpretreatedcontrol, and that saccharification at high dry biomass concentration inthe PEHReactor is very effective in releasing sugars.

Example 14

Pretreatment of Yellow Poplar Sawdust at High Biomass Concentration,High Temperature, and Very Low Ammonia, and Saccharification at Low andHigh Concentration

The PEHReactor, without attrition media, was charged with yellow poplarsawdust (596 g, dry weight basis; purchased from Sawmiller Inc.,Haydenville, Ohio). A vacuum was applied to the reactor vessel, anddilute ammonium hydroxide solution was injected to give an ammoniaconcentration of 6 g/100 g dry weight of biomass and a dry weight ofbiomass concentration of 44 g/100 g total biomass-aqueous ammoniamixture. The reactor vessel charged with ammonia and yellow poplarsawdust was brought to 4° C. as described in Example 13, and rotated at4° C. for 30 min. At this time the contents were transferred to thesteam gun reactor. Once the steam gun reactor was charged with theammonia-poplar mixture, the temperature was increased to 145° C. and themixture was held at temperature for 20 minutes. At the end of thepretreatment time, the yellow poplar sawdust was discharged from thesteam gun reactor through a 1-in circular die into a flash tank. Asample of pretreated yellow poplar sawdust was subsequently saccharifiedas described in Example 13 in a shake flask, and another sample wassaccharified in the PEHReactor. The shake flask saccharification wascarried out at 5% dry weight of biomass relative to the total weight ofthe pretreated biomass-saccharification enzyme consortium mixture, whilethe PEHReactor saccharification (using ˜279 g dry weight pretreatedsawdust) was carried out at 30% dry weight of biomass relative to thetotal weight of the pretreated biomass-saccharification enzymeconsortium mixture. Unpretreated yellow poplar sawdust was alsosaccharified at 5% dry weight of biomass relative to the total weight ofthe pretreated biomass-saccharification enzyme consortium mixture in ashake flask. All saccharifications were done with 28.4 mg/g celluloseSpezyme CP® cellulase and 28.4 mg/g cellulose Multifect® xylanase at 50°C. and pH 5.5 for 96 hr. Yields given in Table 12 below are the releaseor each sugar as a percentage of theoretical yield.

TABLE 12 Yields following pretreatment and saccharification of yellowpoplar sawdust. No pretreatment Pretreated Pretreated 5% DWB 5% DWB 30%DWB Component saccharification saccharification saccharification Monomer2.7% 11.1% 20.6% glucose Total glucose ND ND 30.0% Monomer   0% 17.9%18.9% xylose Total xylose ND ND 40.2% ND: not determined

The results demonstrate that pretreatment of yellow poplar sawdust withvery low ammonia allows substantial sugar release as compared to theunpretreated control, and that saccharification at high dry weight ofbiomass in the PEHReactor is more effective in releasing sugars than theshake flask.

Example 15 Ethanol Production by Yeast Fermentation on Hydrolyzate fromVery Low Ammonia-Pretreated and Saccharified Cob Biomass

The same hydrolyzate used to produce 1,3-propanediol in Example 10 wasalso used to produce ethanol by yeast fermentation. This hydrolyzate wasused as a source of fermentable sugar for conversion to ethanol bywild-type Saccharomyces cerevisiae in shake flasks. The hydrolyzate wasused at 10% (v/v) strength, with the balance being aqueous mediumconsisting of 10 g/L yeast extract, and 20 g/L peptone. Yeast werecultured in 50 mL of medium in a 250 mL baffled flask. The cultures wereincubated at 30° C. with 250 rpm shaking for 24 hours. The amount ofethanol produced was measured by HPLC as described in Example 9, andresults from duplicate flasks are listed in Table 13 below.

TABLE 13 Substrate utilization and product formation by fermentationwith yeast. Broth at time zero Flask 1, 24 hr Flask 2, 24 hr Glucose(g/L) 13.9 1.3 0.9 Ethanol (g/L) 0 4.1 4.3 Glucose use 0 91% 94%

Example 16 Lactic Acid Production by Lactobacillus Fermentation onHydrolyzate from Very Low Ammonia-Pretreated and Saccharified CobBiomass

The same hydrolyzate used to produce 1,3-propanediol in Example 10 wasalso used to produce lactic acid by fermentation with Lactobacillusbrevis in shake flasks. The hydrolyzate was used at 10% (v/v), with thebalance being aqueous medium consisting of 5 g/L yeast extract, 10 g/Lpeptone, 2 g/L ammonium citrate, 5 g/L sodium acetate, 0.1 g/L MgSO₄,0.05 g/L MnSO₄ and 2 g/L K₂HPO₄ and 1 g/L Tween. The Lactobacillus wascultured in 50 mL broth in 250 mL baffled flasks. Duplicate cultureswere incubated at 34° C. with 150 rpm shaking for 24 hours. The amountof lactic acid produced was measured by HPLC as described in Example 10and is listed in Table 14. The two Flask 2 samples are duplicate assaysof the same culture.

TABLE 14 Substrate utilization and product formation by fermentationwith Lactobacillus brevis Broth at time Flask 1, Flask 2, Flask 2, zero24 hr 24 hr 24 hr Glucose (g/L) 25.1 4.6 3.9 3.3 Lactic acid (g/L) 0 8.86.2 6.4 Glucose use 0 82% 84% 87%

Example 17 Cob Pretreatment at Higher Dry Biomass Concentration withVery Low Ammonia

Whole corn cobs were processed with a jaw crusher (2.2 kW motor) with ajaw spacing of approximately 0.95 cm, followed by a delumper (1.5 kWmotor, Franklin Miller Inc., Livingston, N.J.), followed by screeningwith a Sweco screen equipped with a 1.9 cm U.S. Standard screen.Approximately 805 g fractured cobs were loaded into the PEHReactor.Moisture content in the cobs was approximately 7%. The atmosphere in thereactor vessel was flushed 5 times with nitrogen prior to loading. Thereactor, with no attrition media, was preheated to 75° C. before thestart of the experiment, without rotation. When the temperature withinthe reactor vessel stabilized at 75° C. the rolling mechanism in theincubator was turned on and the rotation adjusted to 19 rpm. Theappropriate amount of dilute ammonium hydroxide solution to give anammonia concentration of 6 g ammonia/100 g dry weight of biomass and asolids concentration of 50 g dry weight of biomass/100 g total weight ofbiomass-ammonia mixture was then pumped into the reactor. Ethanol at 1g/100 g dry weight of biomass was also added to the solution. Theammonia solution was pumped through a heated loop in a water bath heatedto ˜75° C., fabricated using a 2-gal Parr reactor. The heated diluteammonium hydroxide solution was injected via an injection lance into thereactor vessel and sprayed on the fractured cobs rotating and tumblingin the reactor. The reactor was maintained at 75° C. for 2 hr whileturning at 19 rpm. At the end of that time, a vacuum (approximately 85kPa) was applied to the reactor vessel for 30 minutes to remove ammoniaand drop the temperature of the contents of the reactor to approximately50° C. Carbon dioxide was then injected into the reactor to relieve thevacuum and the reactor was pressurized to 103 kPa gauge pressure CO₂ andheld at pressure for 30 min at 50° C.

Following this, the reactor was depressurized, opened and attritionmedia were added. The pH of the contents was adjusted to approximately5.5 by injecting 1 M citric acid buffer at pH 4.8 using the injectionlance, to increase the citric acid buffer strength to ˜75 mM, plusadding citric acid monohydrate. Not all of the ammonia was stripped offin the vacuum step nor neutralized with CO₂. The citric acid buffer wasinjected into the reactor following heating to 50° C. and then thecontents was allowed to equilibrate by incubating the reactor at 50° C.and 19 rpm for 1 hour. Injection of the citric acid buffer whilerotating the reactor using the injection lance allowed for a more evenspraying and distribution of the buffer on the pretreated cob particles.The reactor was removed from the incubator, opened, and the pH of asample determined. If the pH was above 5.5, then additional solid citricacid monohydrate was added and the reactor was incubated with mixing at50° C. for an additional hour. This process was repeated until the pHwas approximately 5.5. Once the desired pH was reached, 12.9 mg/gcellulose Spezyme CP (Genencor) and 5 mg active protein/g celluloseenzyme consortium consisting of β-glucosidase, xylanase, β-xylosidaseand arabinofuranosidase were loaded into the reactor. The reactorremained in the incubator at 50° C. and 19 rpm for 72 hr. Following thispretreatment and saccharification, monomer glucose yield was 62.0% andmonomer xylose yield was 31.0%. Total glucose yield was 75.2% and totalxylose was 80.3%.

Example 18

Cob Pretreatment at Higher Solids Concentration with Very Low Ammoniaand Alternate Conditions

Whole corn cobs were processed with a hammermill (10-inch hammer mill,Glen Mills Inc., Clifton, N.H.) to pass through a 1.27 cm screen.Approximately 805 g fractured cobs were loaded into the PEHReactor.Moisture content in the cobs was approximately 7%. Twenty-two ceramicattrition cylinders (3.2 cm diameter×3.2 cm long; E. R. AdvancedCeramics, East Palestine, Ohio) were also added to the reactor. Thereactor was preheated to 95° C. before the start of the experiment,without rotation. A vacuum (approximately 85 kPa) was applied to thereactor vessel before the start and the vessel was sealed off. When thetemperature within the reactor vessel stabilized at 95° C. the rollingmechanism in the incubator was turned on and the rotation adjusted to 19rpm. The appropriate amount of dilute ammonium hydroxide solution togive an ammonia concentration of 6 g ammonia/100 g dry weight of biomassand a solids concentration of 50 g dry weight of biomass/100 g totalweight of biomass-ammonia mixture was then pumped into the reactor. Theammonia solution was pumped through a heated loop in a boiling waterbath fabricated using a 2-gal Parr reactor. The heated dilute ammoniumhydroxide solution was injected via an injection lance into the reactorvessel and sprayed on the fractured cobs rotating and tumbling in thereactor. The reactor was maintained at 95° C. for 2 hr while turning at19 rpm. At the end of that time, a vacuum (approximately 85 kPa) wasapplied to the reactor vessel for 30 minutes to remove ammonia and dropthe temperature of the contents of the reactor to approximately 50° C.Carbon dioxide was then injected into the reactor to relieve the vacuumand the reactor was pressurized to 103 kPa gauge pressure and held atpressure for 30 min at 50° C.

Following this, the reactor was depressurized, opened and the pH of thecontents was adjusted to approximately 5.5 by injecting 1 M citric acidbuffer, pH 4.8, into which citric acid monohydrate was added anddissolved. The citric acid buffer was injected into the reactorfollowing heating to 50° C. and then the contents was allowed toequilibrate by incubating the reactor at 50° C. and 19 rpm for 1 hour.Injection of the citric acid buffer while rotating the reactor using theinjection lance allowed for a more even spraying and distribution of thebuffer on the pretreated cob particles. The reactor was removed from theincubator, opened, and the pH of a sample determined. If the pH wasabove 5.5, then additional solid citric acid monohydrate was added andthe reactor was incubated with mixing at 50° C. for an additional hour.This process was repeated until the pH was approximately 5.5. Once thedesired pH was reached, 12.9 mg/g cellulose Spezyme CP (Genencor) and 5mg active protein/g cellulose enzyme consortium consisting ofβ-glucosidase, xylanase, β-xylosidase and arabinofuranosidase wereloaded into the reactor. The reactor remained in the incubator at 50° C.and 19 rpm for 72 hr. Following this pretreatment and saccharification,monomer glucose yield was 50.7% and monomer xylose yield was 35.7%.Total glucose and xylose yields were 71.7% and 89.8%, respectively.

Example 19

Pretreatment of Cobs with Very Low Ammonia and Additional Base

Whole corn cobs were processed with a jaw crusher (2.2 kW motor) with ajaw spacing of approximately 0.95 cm, followed by a delumper (1.5 kWmotor, Franklin Miller Inc.), followed by screening with a Sweco screenequipped with a 1.9 cm U.S. Standard screen. Approximately 460 gfractured cobs were loaded into the PEHReactor. Moisture content in thecobs was approximately 7%. The reactor was preheated to 95° C. beforethe start of the experiment, without rotation. A vacuum (approximately85 kPa) was applied to the reactor vessel before the start and thevessel was sealed off. When the temperature within the vesselre-stabilized at 95° C. the rolling mechanism in the incubator wasturned on and the rotation was adjusted to 19 rpm. The appropriateamount of ammonium hydroxide solution to give an ammonia concentrationof 3.2 g ammonia/100 g dry weight of biomass and NaOH to give aconcentration of 1.9 g NaOH/100 g dry weight of biomass whilemaintaining a solids concentration of 30 g dry weight of biomass/100 gtotal weight of biomass-ammonia mixture was then pumped into thereactor. The ammonia and additional base solution was pumped through aheated loop in a boiling water bath fabricated using a 2-gal Parrreactor. The heated dilute ammonium hydroxide solution was injected viaan injection lance into the reactor vessel and sprayed on the fracturedcobs rotating and tumbling in the reactor. Following injection, thevacuum on the vessel was relieved to atmospheric pressure. The reactorwas maintained at 95° C. 30 min, then the temperature was lowered to 85°C. where it was maintained for 4 hr. At the end of that time, a vacuum(approximately 85 kPa) was applied to the reactor vessel for 30 minutesto remove ammonia and drop the temperature of the contents of thereactor to approximately 50° C. Carbon dioxide was then injected intothe reactor to relieve the vacuum and the reactor was pressurized to 103kPa gauge pressure and held at pressure for 30 min at 50° C.

-   -   Following this, the reactor was depressurized, opened and the pH        of the contents was adjusted to approximately 5.5 by injecting        approximately 75 ml of 1 M citric acid buffer, pH 4.8, into        which citric acid monohydrate was added and dissolved. The        citric acid buffer was injected into the reactor following        heating to 50° C. and the contents was then allowed to        equilibrate by incubating the reactor at 50° C. and 19 rpm for 1        hour. Injection of the citric acid buffer while rotating the        reactor using the injection lance allowed for a more even        spraying and distribution of the buffer on the pretreated cob        particles. The reactor was removed from the incubator, opened,        and the pH of a sample determined. If the pH was above 5.5, then        additional solid citric acid monohydrate was added and the        reactor was incubated with mixing at 50° C. for an additional        hour. This process was repeated until the pH was approximately        5.5. Once the desired pH was reached, 28.4 mg/g cellulose        Spezyme CP (Genencor) and 28.4 mg/g cellulose Multifect were        loaded into the reactor. The reactor remained in the incubator        at 50° C. and 19 rpm for 72 hr. Following this pretreatment and        saccharification, monomer glucose yield was 56.1% and monomer        xylose yield was 39.5%. Total glucose and xylose yields were        82.8% and 84.2%, respectively. These values are the averages of        2 experiments.

Example 20

Room Temperature and Very Low Ammonia Pretreatment

Whole corn cobs were processed with a jaw crusher (2.2 kW motor) with ajaw spacing of approximately 0.95 cm, followed by a delumper (1.5 kWmotor, Franklin Miller Inc.), followed by screening with a Sweco screenequipped with a 1.9 cm U.S. Standard screen. Approximately 460 gfractured cobs were loaded into the PEHReactor. Moisture content in thecobs was approximately 7%. Twenty-two ceramic attrition cylinders (3.2cm diameter×3.2 cm long; E. R. Advanced Ceramics, East Palestine, Ohio)were also added to the reactor. A vacuum (approximately 85 kPa) wasapplied to the reactor vessel before the start and the vessel was sealedoff. When the temperature within the reactor re-stabilized at roomtemperature (22-26° C.) the rolling mechanism in the incubator wasturned on and rotation was adjusted to 19 rpm. The appropriate amount ofdilute ammonium hydroxide solution to give an ammonia concentration of 4g ammonia/100 g dry weight of biomass and while maintaining a solidsconcentration of 30 g dry weight of biomass/total weight ofbiomass-ammonia mixture was then pumped into the reactor. The diluteammonium hydroxide solution was injected via an injection lance into thereactor vessel and sprayed on the fractured cobs rotating and tumblingin the reactor. Following injection, the vacuum on each vessel wasrelieved to atmospheric pressure. The reactor was maintained at roomtemperature (22-26° C.) for 24 hr. At the end of that time, a vacuum(approximately 81 kPa) was applied to the reaction vessel for 30 minutesto remove ammonia. Carbon dioxide was then injected into the reactor torelieve the vacuum and the reactor was pressurized to 103 kPa gaugepressure with CO₂ and held at pressure for 30 min at room temperature.

Following this, the reactor was depressurized, opened and the pH of thecontents was adjusted to approximately 5.5 by adding citric acidmonohydrate following heating to 50° C., and then allowed to equilibrateby incubating the reactor at 50° C. and 19 rpm. The reactor was removedfrom the incubator, opened, and the pH of a sample determined. If the pHwas above 5.5, then additional solid citric acid monohydrate was addedand the reactor was incubated with mixing at 50° C. This process wasrepeated until the pH was approximately 5.5. Once the desired pH wasreached, 12.9 mg/g cellulose Spezyme CP (Genencor) and 5 mg activeprotein/g cellulose enzyme consortium consisting of β-glucosidase,xylanase, β-xylosidase and arabinofuranosidase were loaded into thereactor. The reactor remained in the incubator at 50° C. and 19 rpm for72 hr. Following this pretreatment and saccharification, monomer glucoseyield was 41.7% and the monomer xylose yield was 25.4%. Total glucoseand xylose yields were 50.1% and 53.2%, respectively. These values werethe averages of 2 experiments.

1. A method for treating biomass to produce fermentable sugarscomprising: a) for a period of time up to about 25 hours, contactingbiomass with an aqueous solution comprising ammonia to form abiomass-aqueous ammonia mixture, wherein the ammonia is present at aconcentration at least sufficient to maintain alkaline pH of thebiomass-aqueous ammonia mixture wherein said ammonia is present at 12weight percent or less, relative to dry weight of biomass, and furtherwherein the dry weight of biomass is at a solids concentration of about15 weight percent or more relative to the weight of the biomass-aqueousammonia mixture, to produce a pretreated biomass product; and b)contacting the product of step (a) with a saccharification enzymeconsortium comprising one or more hemicellulose-hydrolyzing glycosidase,under suitable conditions, to produce fermentable sugars wherein saidfermentable sugars provide a carbohydrate source for a biocatalyst for afermentation process.
 2. The method of claim 1 wherein the pH of thebiomass-aqueous ammonia mixture is greater than
 8. 3. The method ofclaim 1 wherein vacuum is applied to the biomass prior to contacting thebiomass with an aqueous solution comprising ammonia.
 4. The method ofclaim 1 wherein said dry weight of biomass is a solids concentration offrom about 15% to about 80%.
 5. The method of claim 3 wherein said dryweight of biomass is a solids concentration of from about 15% to about60%.
 6. The method of claim 1 wherein said ammonia is present at 10weight percent, or less, relative to dry weight of biomass.
 7. Themethod of claim 1 wherein said ammonia is present at 6 or less weightpercent relative to dry weight of biomass.
 8. The method of claim 1wherein biomass is selected from the group consisting of bioenergycrops, agricultural residues, municipal solid waste, industrial solidwaste, yard waste, wood and forestry waste.
 9. The method of claim 1wherein biomass is selected from the group consisting of switchgrass,waste paper, sludge from paper manufacture, corn grain, corn cobs, cornhusks, corn stover, grasses, wheat, wheat straw, hay, rice straw, sugarcane bagasse, sorghum, soy, trees, branches, roots, leaves, wood chips,sawdust, shrubs and bushes, vegetables, fruits, flowers and animalmanure.
 10. The method of claim 9 wherein biomass is selected from thegroup consisting of corn cobs, corn stover, corn husks, sugar canbagasse, switchgrass, wheat straw, hay, barley, barley straw, ricestraw, and grasses.
 11. The method of claim 10 wherein biomass isselected from the group consisting of corn cobs, corn stover and sugarcane bagasse.
 12. The method of claim 1 wherein the ammonia is selectedfrom the group consisting of ammonia gas, ammonium hydroxide, urea, andcombinations thereof.
 13. The method of claim 1 wherein said aqueoussolution comprising ammonia further comprises at least one additionalbase.
 14. The method of claim 13 wherein said at least one base isselected from the group consisting of sodium hydroxide, sodiumcarbonate, potassium hydroxide, potassium carbonate, calcium hydroxide,and calcium carbonate.
 15. The method of claim 1 wherein (a) is carriedout a temperature of from about 4° C. to about 200° C.
 16. The method ofclaim 15 wherein (a) is carried out a temperature of from about 75° C.to about 150° C.
 17. The method of claim 16 wherein (a) is carried out atemperature of from greater than 90° C. to about 150° C.
 18. The methodof claim 1 wherein (a) is carried out for a period of time of up toabout 8 hours.
 19. The method of claim 1 or claim 2 wherein at least aportion of the ammonia of a) is removed prior to (b).
 20. The method ofclaim 1 wherein ammonia from (a) is recycled.
 21. The method of claim 1wherein the contacting of (b) is at a dry weight of biomassconcentration of at least 15%.
 22. The method of claim 1 wherein (a),(b) or (a) and (b) are repeated at least one time.
 23. The method ofclaim 1 further comprising adding at least plasticizer, softening agentor combination thereof in (a).
 24. The method of claim 23 wherein saidat least one plasticizer, softening agent or combination thereof isselected from the group consisting of polyols, esters of polyols, glycolethers, acetamide, ethanol, and ethanolamines.
 25. The method of claim1, further comprising applying energy before or during (a), before orduring (b), or a combination thereof.
 26. The method of claim 25 whereinsaid energy is selected from the group consisting of milling, crushing,grinding, shredding, chopping, disk refining, ultrasound and microwave.27. The method of claim 1, wherein carbon dioxide from fermentation isused to adjust the pH of the pretreatment mixture prior tosaccharification.
 28. The method of claim 1 wherein saidsaccharification enzyme consortium further comprises at least oneadditional enzyme selected from the group consisting ofcellulose-hydrolyzing glycosidases, starch-hydrolyzing glycosidases,peptidases, lipases, ligninases and feruloyl esterases.
 29. The methodof claim 1 wherein said saccharification enzyme consortium comprises atleast two enzymes selected from the group consisting of cellulases,endoglucanases, exoglucanases, cellobiohydrolasese, β-glucosidases,xylanases, endoxylanases, exoxylanases, β-xylosidases, arabinoxylanases,mannases, galactases, pectinases, glucuronidases, amylases, α-amylases,β-amylases, glucoamylases, α-glucosidases, and isoamylases.
 30. Themethod of claim 1 wherein step (b) is performed at a temperature of fromabout 15° C. to about 100° C. and at a pH of from about 2 to about 11.